PMC

Inhibition of matriptase by identified Fabs. The relative inhibition of matriptase by eight Fabs identified by phage display is shown. The most potent inhibitor A11 was further investigated and fully characterized.

Comparison of the amino acid sequences (Kabat numbering) of the A11, S4, and E2 heavy (H1, H2, H3) and light chain (L1, L2, L3) variable loops.

IC₅₀ plots for the A11, S4, and E2 circularized H3 peptides against matriptase. (a) IC₅₀ of circularized A11 H3 peptide at time zero (red) and 24 hours (blue). The IC₅₀ increases over time due to digestion of the peptide. (b) IC₅₀ of circularized S4 H3 peptide at time zero (red) and 24 hours (blue). The IC₅₀ improves modestly over time. (c) IC₅₀ of circularized E2 H3 peptide at time zero (red) and 24 hours (blue). The IC₅₀ remains the same.

Cartoon model highlighting the reverse orientation of the H3 loops of A11 and S4 at the matriptase active site. (a) BPTI adopts a substrate orientation at the matriptase active site. By comparison, the reverse orientation of the (b) H3 loop of A11 and (c) H3 loop of S4 in the matriptase active site provides an additional factor to prevent cleavage of the H3 loop. (d) H3 loop of E2 also adopts a substrate orientation at the matriptase active site. The catalytic serine residue (OH) and the matriptase substrate pockets (S4, S3, S2, S1, S1’, S2’) are also shown in the schematic.

Matriptase alanine point mutants and their effect on protease inhibition by A11 and S4. (a) The six matriptase surface loops surround the protease active site, which consists of a binding cleft and the catalytic triad (sticks, in yellow). (b) Matriptase surface map of K_I changes with A11. (c) Matriptase surface map of K_I changes with S4. Point mutants that had minimal effect on protease inhibition are shaded in gray, mutations that had a three to tenfold increase in inhibitor K_I are shaded in pink, and point mutants that increased inhibitor K_I by more than tenfold are shaded in red. Point mutants that decreased inhibitor K_I are shaded in green. The point-mutant/inhibitor K_I values are given in . Structural figures were generated using PyMOL (Schrodinger LLC).

Close-up view of the H3 loop in the (a) A11/matriptase and (b) S4/matriptase complexes. The Fab (heavy chain, light pink; light chain, light orange) caps matriptase surface loops (space-filling models) with the 60s loop shaded (red), 37s loop (purple), 140s loop (orange), 220s loop (dark pink), 170s loop (light blue) and 90s loop (green) as indicated. The H3 loops (dark blue) of A11 and S4 are inserted directly into the active site of matriptase (catalytic triad indicated in yellow) while the remaining variable loops (L1, L2 and L3, red; H1 and H2, sky blue) interact with the protease surface loops. Structural figures were generated using PyMOL. (c) Buried surface area as determined by PISA shows the contribution of the heavy and light chains to the antibody-matriptase interactions. The number in parentheses represents the % of the total buried surface area.

Interaction of the H3 variable loops of A11 and S4 with the matriptase substrate-binding pocket. (a) The H3 loop of A11 (blue) accounts for the majority of the buried surface area contributed by the heavy chain variable loops. The loop inserts ArgH100b into the active site at the S1 pocket while making very few additional contacts. A11 is superimposed with chloromethylketone inhibitor D-FPR-cmk (purple, PDB code 2FIR). (b) The H3 loop of S4 (green) inserts into the matriptase substrate-binding pocket and makes contacts with a number of the matriptase residues. S4 is superimposed with D-FPR-cmk (purple). (c) ArgH100b makes a water-mediated hydrogen bond with Asp189. Benzamidine (red, ball and stick, PDB code 1EAX) bound to matriptase adapts the expected binding orientation for an arginine containing substrate, forming a salt bridge with Asp189 at the bottom of the S1 pocket of matriptase (distance of 3.1 Å). H3 loop of A11 in blue and H3 loop of S4 in green. (d) Residue ArgH99 of S4 is a critical binding residue for matriptase inhibition and ArgH100b of S4 makes the same water-mediated hydrogen bond with Asp189 as A11.